Methods for Measuring Changes in Optical Properties of Wound Tissue and Correlating Near Infrared Absorption (FNIR) and Diffuse Reflectance Spectroscopy Scattering (DRS) With Tissue Neovascularization and Collagen Concentration to Determine Whether Wound is Healing

ABSTRACT

Optical changes of tissue during wound healing measured by Near Infrared and Diffuse Reflectance Spectroscopy are shown to correlate with histologic changes. Near Infrared absorption coefficient correlated with blood vessel in-growth over time, while Diffuse Reflectance Spectroscopy (DRS) data correlated with collagen concentration. Changes of optical properties of wound tissue at greater depths are also quantified by Diffuse Photon Density Wave (DPDW) methodology at near infrared wavelengths. The diffusion equation for semi-infinite media is used to calculate the absorption and scattering coefficients based on measurements of phase and amplitude with a frequency domain or time domain device. An increase in the absorption and scattering coefficients and a decrease in blood saturation of the wounds compared to the non wounded sites was observed. The changes correlated with the healing stage of the wound. The methodologies used to collect information regarding the healing state of a wound may be used to clinically assess the efficacy of wound healing agents in a patient (e.g., a diabetic) and as a non-invasive method

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority to U.S. Provisional PatentApplication No. 61/046,640, filed Apr. 21, 2008, and to U.S. ProvisionalPatent Application No. 61/054,535, filed May 20, 2008. The contents ofthese patent applications are hereby incorporated by reference in theirentireties.

FIELD OF THE INVENTION

The invention relates to methods for measuring changes in opticalproperties of tissue during acute wound healing and, more particularly,to the use of diffuse photon density wave (DPDW) methodology at nearinfrared frequencies to calculate the absorption and scatteringcoefficients of wound tissue based on measurements of phase and/oramplitude with a continuous wave, a frequency domain, or a time domainnear infrared device. The invention also relates to determining whethera wound is healing by assessing tissue neovascularization and collagenconcentration in a wound by correlating measurements made using nearinfrared absorption and diffuse reflectance spectroscopy scattering andby monitoring changes in oxygenated hemoglobin over time. The inventionapplies to subsurface tissue optical properties and oxygenation andrelates to ischemic environments, impaired healing states, and emergingwounds such as pressure or ubiquitous ulcers.

BACKGROUND OF THE INVENTION

Assessment of healing in chronic wounds is gaining importance as new andexpensive wound treatments are brought to market. A wide variety ofchronic wound treatments such as topical growth factors, bioengineeredskin equivalents, negative pressure wound therapy, and hyperbaric oxygentherapy are commercially available and clinical studies of theseproducts have shown some evidence of improved healing compared tostandard of care. However, the effectiveness of each treatment is notthe same in all patients, so rapid and accurate evaluation of healingprogress in each individual is critical so that unsuccessful treatmentscan be discontinued and alternate treatments initiated as soon aspossible. Reliable methods of evaluating wound healing would benefitboth wound clinics by reducing the duration and cost of treatment, andthe wound research community in the evaluation of clinical trials.

The main limitation of traditional wound evaluations is that they cangive information mostly from the surface of the wound. Such surfacecharacteristics of a wound do not take into account the health of thewound environment beneath the surface in the whole wound bed, andprovide inadequate information regarding the wound healing status of awound. Therefore, misdiagnosis may occur or treatment may not be alteredas early as possible, with direct implications on the quality and costof care for chronic wounds. For example, image analysis of woundpictures for color or texture pertains strictly to surface informationand optical methods such as Diffuse Reflectance Spectroscopy (DRS) orOptical Coherence Tomography (OCT) can penetrate to only approximately 1millimeter. Non-invasive analysis of the full depth of the chronic woundbed could provide the clinician with a more complete picture of woundhealth, allowing better prediction of wound closure and wound recurrencethan can be achieved by surface measurements alone.

Several human studies have been conducted in an attempt tonon-invasively characterize tissue beneath the surface of chronicwounds. High frequency ultrasound (HFUS) at frequencies in the range of20 MHz permits high resolution (microscopic-level) imaging of skin atdepths of up to 2 cm. A preliminary study showed that HFUS could be usedto image structural features beneath the surface of human chronic woundsand qualitative comparisons were made with healthy skin. HFUS was usedto measure skin thickness in several types of human chronic wounds(diabetic, venous, pressure, and ubiquitous ulcers), and a later studyby Dyson et al. described in “Wound healing assessment using 20 MHzultrasound and photography,” Skin Research and Technology, 2003, Vol. 9,pages 116-121, demonstrated the use of HFUS to calculate the width anddepth of small acute wounds that were created experimentally in humansubjects. However, it is unclear how this method would translate tochronic wounds that are very different in shape, size, and also havemore ambiguous boundaries than acute wounds.

Optical Coherence Tomography (OCT) is a non-invasive imaging modalitythat uses low coherence interferometry to create high resolutioncross-sectional images of structural features in human skin at depths ofup to 1.2 mm. This method has not yet been used to image human wounds,but structures visible in OCT images of experimentally-created animalwounds have been qualitatively correlated to histological micrographs ofthe same wounds, and an automated imaging algorithm was developed tocalculate the size of these acute animal wounds. In another animalstudy, polarization-sensitive OCT was used to monitor temporal changesin collagen birefringence during healing, and measurements ofbirefringence were shown to be greater in chemically accelerated woundhealing as compared to chemically impaired healing. As with HFUS, theclinical utility of OCT as a wound monitoring methodology is uncertaindue to the size and complexity of human chronic wounds.

Laser Doppler Flowmetry (LDF) and its modified methodology of LaserDoppler Imaging (LDI) are optical methods that rely on frequency shiftsof an incident light beam (typically a laser in the near infraredwavelength range) to determine a quantitative index that is related tothe average velocity and number of red blood cells within a tissuevolume. Some researchers have used LDF and LDI to quantify relativevalues of cutaneous blood flow in human chronic wounds. These studiesidentified regions of increased blood flow within chronic wounds thatmay correlate to granulation tissue; however, changes in blood flow werenot monitored over time. The clinical utility of LDF and LDI for serialassessment of chronic wounds is limited due to low penetration depths(˜1-2 mm) and issues with light reflection caused by curvature of thefeet and presence of moisture on the surface of the wound.

Diffuse Reflectance (or Remittance) Spectroscopy (DRS) is an opticalmethod that uses light at visible and near infrared wavelengths (400 to1500 nm) to measure hemoglobin concentration and oxygenation of blood insuperficial capillaries, to depths of approximately 1 mm. DRS spectrafrom chronic leg ulcers (both venous and arterial) have been empiricallycorrelated to qualitative wound scores assessed by physicians, andchanges in oxygen saturation were measured over the course of healingusing DRS in diabetic foot ulcers. However, changes of the surfaceappearance due to bleeding and other reasons will significantly affectthe capability of DRS to provide on its own information about the woundstatus and oxygenation.

Generally speaking, the determination of wound surface area is highlyinaccurate and subjective. (See Robson, M. C., et al., “Wound HealingTrajectories as Predictors of Effectiveness of Therapeutic Agents,” inArchives of Surgery. 2000, Am Med. Assoc. p. 773-777). Wound edges maybe hard to determine because of complex wound geometry. Width and depthmeasurements may vary from between observers during the same clinicsession and are highly inaccurate between visits. Surface area does nottake into account changes in wound volume. Ultrasound measurements andimage analysis of digital photos provide more accurate information butare difficult to use in a busy clinical setting.

In previous publications of the present inventors, it has been reportedthat near infrared optical measurements correlated with wound areareduction and were able to distinguish between a diabetic wound and anon-diabetic wound in a rat model. Weingarten, M. S., et al.,“Measurement of optical properties to quantify healing of chronicdiabetic wounds,” Wound Repair and Regeneration, 2006, Vol. 14(3): pp.364-370. As will be explained herein, the inventors have expanded uponthis research by combining Near Infrared (NIR) with Diffuse Reflectancespectroscopy (DRS) and reporting whether the near infrared absorptioncoefficient correlates with histological changes in the wounds andwhether the DRS scattering function correlates with collagenconcentration in the healing tissue.

Moreover, it is established that wounds, burns and lesions need oxygento heal and that ischemic conditions represent impaired healingenvironments. Therefore, by measuring oxygenated hemoglobin,deoxygenated hemoglobin, and oxygen saturation, the inventors suggestthat it is possible to predict wound healing. Current methods inclinical wound care practice rely on estimates of the surface area bymeasuring length and width of the lesion. These methods are highlysubjective and more importantly cannot assess the probability of woundhealing in impaired environments, such as in chronic wounds due todiabetes, venous ulcers, pressure ulcers, ubiquitous ulcers, and others.Invasive monitoring based on biopsies could provide information aboutthe physiology and biochemistry of healing but is invasive andimpractical, while monitoring based on wound fluid is controversial dueto debates over appropriate correlation of wound fluid composition towound tissue.

At present, various optical methods have been proposed and can be usedfor determining parameters representing skin injury or for monitoringthe healing processes. Most optical methods are non-invasive andrelatively inexpensive and as such offer major advantages compared toinvasive methods. Different modifications of diffuse reflectancespectroscopy (DRS) have become the most common methodology in monitoringwounds, burns and lesions. DRS has been used extensively for evaluatingskin changes at superficial depths up to 1 mm because with a typicalbroad range wavelengths source of incident light (400-1500 nm) thestrong absorption exhibited by the tissue inhibits optical probing ofdeeper layers. Using specialized algorithms to fit DRS re-emissionspectra to phantoms and model systems, many investigators obtainedimportant information about the depth of burn injuries, sun damage,topical drug delivery, and water content of the skin.

In wound characterizations, the absence of significant depth penetrationmakes DRS data difficult to interpret. For example, DRS data from asignificant number of wounds had to be collected in order to develop anempirical algorithm that could mimic a clinical wound assessment scorewhich averages clinical observations. In order to probe deeper tissuedepths with optical non-invasive methods, a different approach than DRSis desired. Such an approach is described herein.

SUMMARY OF THE INVENTION

The inventors have found that changes in blood vessel in-growth and/orischemia in a wound may be quantified using near infrared (NIR)measurements and that the collagen concentration may be quantified usingdiffuse reflectance spectroscopy (DRS) measurements in the wound tissue.The NIR scattering coefficient was found to not correlate with collagenconcentration or cell count, but to correlate to vessel organization,possibly due to the depth of tissue probed. Because of the penetrationdepth and the wavelength used in DRS, blood vessel organization andpresence of neutrophils is less a factor in DRS scattering as comparedto collagen. These two methods therefore were found to providecomplementary information.

Differences in the change of the absorption coefficient during the woundhealing period were found, and in control wounds, the rate of change inabsorption coefficients was consistently higher at 685 nm and 830 nmcompared to impaired (e.g., diabetic) wounds. This is the exact behaviorpredicted by vessel in growth in the control as blood vessel growth inthe control proceeded more rapidly. The scattering function determinedby DRS was also found to correlate very well with collagen concentrationdetermined by trichrome staining in both the impaired and controlwounds.

In part to take advantage of these observations, a method of collectinginformation regarding the healing state of a wound is provided. In anexemplary embodiment, the method includes illuminating wound tissue withlight from a light source, measuring the amplitude and/or phase shift ofthe light as it propagates through the wound tissue, calculating anoptical absorption coefficient and/or a reduced scattering coefficientusing the measured values, and correlating collagen concentration in thewound tissue with the reduced scattering coefficient calculated frommeasured parameters and/or correlating blood vessel in-growth and/orischemia in the wound tissue with the optical absorption coefficientusing the measured values. Changes in collagen concentration over timemay be determined from changes in the reduced scattering coefficientover time. Similarly, changes in blood vessel in-growth and/or ischemiaover time may be determined from changes in the optical absorptioncoefficient over time. The light is preferably provided by a laser andtransmitted at a near infrared wavelength such as of 650-870 nm. Thelight may also be transmitted at specific near infrared wavelengths suchas 685 nm, 780 nm, 830 nm, and/or 950 nm. The light output by the lasermay be modulated to produce a diffuse photon density wave (DPDW) in thewound tissue. On the other hand, the light may be used to differentiatean impaired wound (e.g., chronic wounds such as diabetic, pressureulcer, venous ulcer, ubiquitous ulcer, and ischemic wounds) from anon-impaired wound (normally healing wound) by measuring changes inblood vessel in-growth and/or ischemia in the wound over time andcorrelating optical absorption coefficients obtained from the wound overtime with blood vessel in-growth and/or ischemia seen histologically forimpaired and control wounds. In embodiments, the method may includedetecting pressure ulcers or venous ulcers in the wound from changes inthe optical absorption coefficients over time.

The method described herein further includes measuring the size of thewound by calculating wound surface area and measuring a healing rate ofthe wound by calculating the difference between the surface area of thewound at different points in time and dividing the difference by theoriginal surface area of the wound.

In an exemplary embodiment, correlating the collagen concentration inthe wound tissue with the reduced scattering coefficient includescorrelating an increase in a diffuse reflectance spectroscopy scatteringfunction obtained over time in the wound with an increase in collagenduring healing of the wound. In an exemplary embodiment, the collagenconcentration in the wound may be measured using DRS measurements overtime.

The illuminating and measuring steps may be performed using a continuouswave, frequency domain, or time domain measurement device that does notcontact the wound. In this embodiment, the calculation of the absorptionand/or reduced scattering coefficients and a quantification of bloodoxygenation is performed using a diffusion equation for semi-infinitemedia.

In accordance with another aspect of the method, monitoring changes inoxygenated hemoglobin over time provides as an indication of whether thewound is healing. The changes in oxygenated hemoglobin may be quantifiedby calculating a rate of change and variability in optical absorptioncoefficient and hemoglobin concentration over time.

BRIEF DESCRIPTION OF THE DRAWINGS

The above-mentioned and other features and advantages of the inventionwill become apparent from the following detailed description inconnection with the accompanying drawings, of which:

FIG. 1 illustrates the rate of wound healing in diabetic and controlrats, as calculated from image analysis of digital photographs of thewounds.

FIG. 2 illustrates wound contraction in diabetic and control rats, ascalculated from image analysis of digital photographs of the wounds.

FIG. 3 illustrates the average absorption coefficient (μ_(a)) indiabetic non-wound tissue, diabetic wounds, healthy non-wound tissue,and healthy wounds at (a) 685 nm, (b) 780 nm, (c) 830 nm, and (d) 950nm.

FIG. 4 illustrates the percent change of absorption coefficients (μ_(a))at 685 nm in diabetic vs. control wounds, showing that similar resultswere obtained for all wavelengths.

FIG. 5 illustrates the average reduced scattering coefficient (μ_(s)′)in diabetic non-wound tissue, diabetic wounds, healthy non-wound tissue,and healthy wounds at (a) 685 nm, (b) 780 nm, (c) 830 nm, and (d) 950nm.

FIG. 6 illustrate the status of wound healing whereby on Day 5 and Day10 a significantly higher number of neutrophils in the diabetic woundsare present, which was not as apparent on Day 21, although on Day 21there was more organized tissue in the control wounds.

FIG. 7 illustrates lectin staining of the wound demonstrating decreasedvascularity in the diabetic wounds compared to the control wounds.

FIG. 8 illustrates image analysis of DAPI-stained tissue samples for atotal number of cells per image.

FIG. 9 illustrates the ratio of collagen concentration in a control vs.diabetic wound as determined by image analysis of trichrome stainedtissue specimens.

FIG. 10 illustrates a DRS scattering function (arbitrary units) vs.relative collagen concentration as determined by image analysis oftrichrome stained tissue specimens, showing a strong correlation betweenthe DRS scattering function and collagen concentration found for both(a) control rats and (b) diabetic rats.

FIG. 11 illustrates trichrome staining for blood vessels.

FIG. 12 illustrates a timeline of animal studies performed by theinventors for measuring wound healing.

FIG. 13 illustrates the probe placement locations (dark rectangles) inan animal model, where each animal was wounded on the left dorsum andmeasurements were performed on (1) the center of the wound, (2) the edgeof the wound, and (3) healthy tissue on the right dorsum, symmetric tothe wound location.

FIG. 14 illustrates the daily average values of (a) μ_(a) and (b) μ′_(s)in a silicone optical phantom over a 50-day period where each pointrepresents the average of measurements taken on the same day and solidlines represent average values for the entire measurement period.

FIG. 15 illustrates the average absorption and scattering coefficientsfor all animals measured as a function of time, including baselinevalues of (a) left dorsal μ_(a), (b) right dorsal μ_(a), (c) left dorsalμ′_(s), and (d) right dorsal μ′_(s) at 685 nm from study 1 and study 2.

FIG. 16 illustrates left dorsal baseline values of (a) μ_(a) and (b)μ′_(s) from three representative rats where each point represents theaverage of three measurements and error bars represent the standarddeviation.

FIG. 17 illustrates a normalized wound area as a function of healingtime for rats in study 2 where each point represents the average of allrats (n=12) and error bars represent the standard deviation.

FIG. 18 illustrates (a) μ_(a) and (b) μ′_(s) at 685 nm during woundhealing (average ±standard deviation) for animals in study 1 and (c)μ_(a) and (d) μ′_(s) at 685 nm during wound healing (average ±standarddeviation) for animals in study 2.

FIG. 19 illustrates lectin-stained images of wound tissue on (a) day 5,(b) day 10, and (c) day 21 after wound surgery.

FIG. 20 illustrates mean±standard deviation of oxyhemoglobin [HbO₂],deoxyhemoglobin [Hb], and total hemoglobin [HbO₂+Hb] during woundhealing for animals in study 2.

FIG. 21 illustrates oxygen saturation during wound healing for animalsin study 2.

FIG. 22 illustrates a hypothesized clinical wound healing curve comparedagainst the result of the animal study shown in FIG. 18.

FIG. 23 illustrates two-tailed, unpaired t-tests used to compare theaverage optical coefficients at each time point to the average opticalcoefficients on day 3 where the resulting p-values are shown as afunction of time. (a) μ_(a) at center of wound, (b) μ_(a) at edge ofwound, (c) μ′_(s) at center of wound, and (d) μ′_(s) at edge of wound.

FIG. 24 illustrates daily average values of μ_(a) in a silicone opticalphantom over a 61-week period.

FIG. 25 illustrates the measurement locations for a typical diabeticfoot ulcer in the human experiments.

FIG. 26 illustrates plots of μ_(a) at all wavelengths during the courseof the study for a typical healing wound.

FIG. 27 illustrates plots of μ_(a) for a typical non-healing wound.

FIG. 28 illustrates plots of μ_(a) for a unique case in which the woundinitially appeared to be healing, decreasing in size from 31.5 cm² to1.6 cm² over 17 weeks but never closed completely and surgicalintervention was required after week 25, increasing the wound size to17.3 cm².

FIG. 29 illustrates the calculated values of total hemoglobinconcentration [Tot Hb] for the wounds in FIGS. 26-28.

FIG. 30 illustrates the slopes calculated from the optical absorptiontrend lines.

FIG. 31 illustrates the slopes calculated from the hemoglobinconcentration trend lines.

FIG. 32 illustrates the mean rates of change in healing and non-healingwounds compared for all optical absorption coefficients (μ_(a) at 685nm, 780 nm, and 830 nm) and all hemoglobin concentrations ([Tot Hb],[HbO₂], and [Hb]).

FIG. 33 compares the mean values of normalized RMSD in healing andnon-healing wounds using the optical absorption at each wavelength andall hemoglobin concentrations (oxy, deoxy and total).

FIG. 34 illustrates the results of a study of wound healing in sevenobese Zucker Diabetic Fatty (ZDF) rats in which the wounds weremonitored using DPDW methodology throughout the healing cycle and for 42days after wound closure.

FIG. 35 illustrates a hypothesized model of the optical changes observedduring healing.

FIG. 36 illustrates the rate of temporal change of [Tot Hb] in eachwound calculated by fitting the data from the first 10 weeks ofmeasurements to a linear trend line, where the slopes of the 10-weektrend lines are compared to the slopes calculated from all availabledata.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

A detailed description of illustrative embodiments of the presentinvention will now be described with reference to FIGS. 1-36. Althoughthis description provides a detailed example of possible implementationsof the present invention, it should be noted that these details areintended to be exemplary and in no way delimit the scope of theinvention.

Monitoring Surface of Wound to Collect Data Regarding Healing State ofWound Material and Methods

A frequency domain diffuse optical tomography instrument developed bythe School of Biomedical Engineering at Drexel University was used tonon-invasively measure the optical properties of tissue at depths up toseveral millimeters. The instrument includes four laser diodes (685,785, 830, and 950 nm) controlled by an optical switch, four avalanchephotodiode detector channels, and a radio-frequency (RF) generator thatmodulates the laser output at a frequency of 70 MHz. The device measuresthe amplitude and phase shift of light as it propagates through tissue,and uses a diffusion-based model to calculate the optical absorptioncoefficient (μ_(a)) and reduced scattering coefficient (μ′_(s)). A fiberoptic probe delivers light through a single optical fiber and collectslight through four optical fibers spaced at distances of 4 mm to 16 mmfrom the source fiber. The instrument was calibrated and its stabilitydetermined using intralipid solutions of varying concentration andtherefore varying scattering and absorption coefficients. During thedescribed measurements, the instrument was calibrated with solidphantoms. Details of this instrument have been published previously byWeingarten, M. S., et al. in “Measurement of optical properties toquantify healing of chronic diabetic wounds,” Wound Repair andRegeneration, 2006, Vol. 14(3): pp. 364-370. As those skilled in the artwill appreciate, chromophores in the wound that absorb light deliveredat these wavelengths are primarily oxy and deoxyhemoglobin and water.

Experimental Animal Model:

An animal model consisting of hairless rats, made diabetic byintraperitoneal Streptozotocin (STZ) administration, was used. Inparticular, the animal model chosen was the hairless female rat. Duringthe course of the study, animals were housed in individual cages onalpha cellulose bedding and maintained in an animal care facility with a12 hour light and dark cycle. Food and water were supplied ad libitum.

Thirty, ten week old Sprague-Dawley female hairless rats, weighingapproximately 205 g, were acquired. Baseline near infrared data werecollected on all rats for 14 days. Eighteen rats were rendered diabeticusing intraperitoneal injection of STZ at 75 mg/kg. Twelve rats weremaintained as the control group. In order to assure successful inductionof diabetes, blood glucose levels were monitored in diabetic rats. Onday 36, a full thickness wound of 4.6 cm² was made using steriletechnique in an animal surgical suite. One wound was inflicted on theleft side of the dorsal area of each animal. The right side of eachanimal was left unwounded to provide a control site and to enableassessment of any systemic changes in optical properties connected toeither diabetes or the wound. The surgery was performed using isofluraneanesthesia administered via a face mask. All wounds were covered with aTegaderm (3M, Minneapolis, Minn.) sterile transparent dressing. Aftersurgery all rats were fitted with “Elizabethan” type collars to preventthem from scratching their wounds. Blood sugar and weight were checkedweekly in the rats.

Optical Measurement of Wound

Near Infrared Spectroscopy (NIR)

NIR optical measurements were performed on 2 locations of the wound side(center of the wound and peri-wound), and on one location on the control(right) side. Before measuring, pooled blood or fluid in the wound wasremoved with gauze. Optical data were collected from the peri-woundarea, the wound center, and the symmetrical unwounded right side(control side). Each position was measured three times to ensurereproducibility. The data reported reflect the average of these threemeasurements and standard error was less than 2%. Measurement of thewounds using the near infrared instrument was performed twice weekly.

The optical device was calibrated before each experiment. The choice ofwavelengths including 680-870 nm allowed assessment of the predominantchromophores in the wound oxy and deoxyhemoglobin. The addition of the950 nm wavelength allowed determination of water concentration in thewound and therefore the state of dehydration of the tissues. The laserswere modulated at 70 MHz to produce a diffuse photon density wave (DPDW)in the tissue. Appropriate algorithms convert the amplitude and phase atthese four wavelengths into measurement of tissue absorption andscattering. Since tissue is a very strong scattering medium with lightbeing scattered at every 1 mm of tissue, an approximation of DPDW wasused to calculate the coefficients of absorption and scattering. Theprobe interrogated the tissue at a depth of approximately 3-5 mm.

Diffuse Reflectance Spectroscopy (DRS)

A diffuse reflectance spectroscopy (DRS) instrument was used to measurethe intensity of backscattered light at a depth between 100-300 micronsfrom the skin/wound surface. The instrument consisted of a Tungstenlight source (Ocean Optics, Boca Raton, Fla.), a bifurcated fiber bundle(Multimode Fiber Optics, East Hanover, N.J.), a spectrophotometer (OceanOptics, Boca Raton, Fla.) and an analyzer. Light was delivered to theskin by one leg of the fiber bundle connected to the light source andcollected by the other leg connected to the spectrophotometer. A fiberoptic probe consisting of 600 randomly mixed optical fibers with 50 μmcore diameter was slightly placed on the skin. The total probe size isabout one half inch in diameter and the active area of the probe isabout 2 mm in diameter. A reflectance spectrum was acquired between 400nm and 750 nm. The DRS scattering function was calculated by finding theintercept at 630 nm of a straight line fitted to the intensity databetween 630 nm and 700 nm using a linear least squares fittingalgorithm. A similar function was used by Knoefel, W. T., et al. in“Reflectance spectroscopy of pancreatic microcirculation,” Journal ofApplied Physiology, 1996, Vol. 80(1), pp. 116-123, to represent ameasure of the scattering intensity.

In vitro measurements of collagen phantoms were performed with DRS toassess the sensitivity of the method in determining collagenconcentration. Three collagen type I gel phantoms, each approximately 1cm thick were made. Collagen gels were prepared from rat tail type Ihigh concentration collagen (BD science, CA) in standard 6-well platesby following the recommended manufacturer's protocol. Briefly, collagenwas dissolved in water to the desired concentration (3 mg/ml, 4.5 mg/ml,and 6 mg/ml). Phosphate buffered saline and 1N NaOH were added toprovide physiological pH and ionic strength. The collagen was allowed togel at 37 degrees C. for about 20-30 minutes.

Determination of Wound Size

Measurements of wound size were determined by calculating wound surfacearea. This was determined using cross polarization digital photographstaken at the same time the near infrared data were collected. The imageanalysis tool IMAGE PRO (Media Cybernetics, Silver Spring, Md.) was usedto calculate the area of each wound. As these wounds were of uniformdepth, wound volume was not calculated. Near infrared spectroscopy, DRS,and digital photography were performed with the rats receiving inhaledisoflurane anesthesia so as to avoid motion artifacts.

Wound Biopsies

With the rats receiving inhaled isoflurane anesthesia, rats in thediabetic group and in the control group had complete excision of theirwounds and the area of the dorsum contralateral to the wound on day 5and 10, and 21 after wounding. These rats were then sacrificed. Woundexcision was performed on 3 control rats and 6 diabetic rats on day 5,and 3 control rats and 6 diabetic rats on day 10. Excisional biopsieswere performed on 6 control rats and 4 diabetic rats on day 21. A totalof 28 wounds and 28 control areas were excised and examinedhistologically.

Hematoxylin and eosin staining was performed in order to observe tissuestructure and cell morphology. Briefly, after rehydration, slides areimmersed in Hematoxylin solution for 3 minutes, and then washed with tapwater for 5 minutes, immersed with Eosin solution for 1 minute anddehydrated with xylene.

Lectin staining (a sugar binding protein of non-immune origin thatagglutinates cells or precipitates glycoconjugates) was used to stainvessels in the tissue, and visualize vascularization. Lectin can be usedas a marker of angiogenesis because it binds to endothelial cellsreveals the overall vascular architecture. Briefly, sections were washedin 1×PBS for 10 minutes after rehydration. Sections were stained withAlexa Fluor 488 conjugated lectin (Invitrogen L2-1415) for 30 minutes inthe dark with a concentration at 1:250 and washed with 1×PBS 3 times for5 minutes each. Determination of microvessel density was performed asdescribed by Weidner et al in “Tumor angiogenesis andmetastasis—correlation in invasive breast carcinoma,” in New EnglandJournal of Medicine, 1991, pp. 1-8. Vessel counts were assessed by lightmicroscopy in areas of the wound tissue containing the highest number ofpositive lectin areas visualized at low power. The six highest areas ofvascularity which did not overlap were identified, a vessel countperformed, and the average of the six counts calculated.

The same procedure as that followed for lectin staining was used forDAPI (4′,6-diamidino-2-phenylindole) (visualize nuclear DNA) but with anadditional step. This step involved mounting sections with VECTASHIELD®and DAPI Mounting Medium. Vessels were stained as fluorescent green andcell nuclei were stained as fluorescent blue. Image analysis of DAPIstained fluorescence images (3-5 images per sample) was performed inorder to assess the number of cells, as described by Otto, F. in “DAPIstaining of fixed cells for high-resolution flow cytometry of nuclearDNA,” Methods Cell Biol, 1990. Col. 33, pp. 105-10.

Collagen fibers were visualized by Gomori's trichrome staining method asdescribed by Gomori, G. in “Aldehyde-fuchsin: a new stain for elastictissue,” Am J Clin Pathol, 1950, Vol. 20(7), pp. 665-6. Trichrome is anacidic dye that selectively stains collagen and is the standard methodused in pathology labs. The image analysis software Image Pro was usedto determine the concentration of collagen by counting the pixelintensity of collagen in a given area.

Results

Wound Size

In the 30 wounds measured during the 21 day period after wounding, woundsize in the control group decreased at a faster rate when compared tothe diabetic group (FIG. 1). Healing rates were calculated according theformula:

Percent Healing=(Original wound area−wound area)/(original wound area)

A statistically significant (p<0.05) difference between the percenthealing of diabetic and control rats was found using the Student t-test.These results duplicated the healing rates observed by this group inWeingarten, M. S., et al., “Measurement of optical properties toquantify healing of chronic diabetic wounds,” Wound Repair andRegeneration, 2006, Vol. 14(3), pp. 364-370.

Wound contraction, was defined as:

Wound contraction=(wound area)/(original wound area).

As illustrated in FIG. 2, wound contraction in the control groupoccurred at a faster rate than the diabetic.

Near Infrared Absorption Data (μ_(a))

Absorption coefficients increased in the diabetic rats starting soonafter the induction of diabetes. Absorption coefficients increasedwithin days of wounding in the diabetic wounds compared to the controls.As illustrated in FIG. 3, the average absorption coefficients weresignificantly higher in the diabetic wounds when compared to thediabetic non-wounded side and to the controls over the time of healing.

As illustrated in FIG. 4, the percent change in absorption coefficients(μ_(a)) in the control wounds was greater than the percentage change inμ_(a) in the diabetic wounds starting from the time of wounding. Percentchange was calculated using the following formula:

${\% \mspace{14mu} {change}} = \frac{\mu_{a} - \mu_{a}^{init}}{\mu_{a}^{init}}$

where μ_(a) ^(init)=μ_(a) from wound on 3rd day after wound surgery (Day3 was the earliest wound measurement). Similar results were obtained forall wavelengths.

Near Infrared Scattering Data (μ_(s))

Scattering coefficients also increased in the diabetic rats startingsoon after the induction of diabetes. As illustrated in FIG. 5, theaverage scattering coefficients were significantly higher in thediabetic wounds when compared to the diabetic non-wounded side and tothe controls soon after wounding and over the time of healing.

FIG. 6 illustrate the status of wound healing where on Day 5 and Day 10a significantly higher number of neutrophils in the diabetic wounds arepresent. This was not as apparent on Day 21; however, on Day 21 therewas more organized tissue in the control wounds. In FIG. 6, a scale barrepresents 50 μm.

FIG. 7 illustrates using lectin staining that the wound demonstrateddecreased vascularity in the diabetic wounds compared to the controlwounds. In FIG. 7, a scale bar represents 25 μm.

An image analysis of the specimens stained with DAPI found that thecontrol wounds have more cells at Day 10 consistent with the normalwound healing processes; at day 5 cell counts could be mostly dominatedby neutrophils, explaining the higher counts seen in the diabetics (FIG.8). This is consistent with impaired wound healing in this model. Therewas no statistical correlation between the μs′ from NIR and Cell countsfrom the DAPI image analysis, because scattering is affected both bycells and collagen.

Trichrome Staining for Collagen

Relative collagen concentration was calculated by image analysis oftrichrome stains of tissue. Collagen concentration was decreased in thediabetic wounds when compared to the control wounds over time, as shownin FIG. 9.

DRS Data

As illustrated in FIG. 10, collagen correlated with the DRS scatteringfunction. As also illustrated in FIG. 10, the DRS scattering functionobtained over time in the diabetic and control wounds also correlatedwith the increase in collagen observed during healing. As illustrated inFIG. 11, the diabetic wound had lower blood vessel ingrowth as predictedby the absorption coefficient data.

Discussion

The experiments described above demonstrate that near infraredspectroscopy may be used to gather data suitable to differentiate therate of normal wound healing from impaired (delayed) healing in ananimal model. Rising values of absorption coefficients at 685 nm, 785nm, and 830 nm during normal wound healing suggested that blood volumewas increasing as blood vessel ingrowth progressed. There was also amarked difference in scattering coefficients in the diabetic wound,suggesting a connection to the number of inflammatory cells orcorrelating to a decreased collagen concentration. The scatteringcoefficient may also be a function of collagen or blood vesselorganization. The NIR scattering coefficient in this model does notcorrelate with collagen concentration or cell count, but it doescorrelate to vessel organization, possibly due to the depth of tissueprobed.

Diffuse Reflectance Spectroscopy (DRS) is a noninvasive optical methodthat provides quantitative information about the structure andcomposition of the superficial 500 μm at most of a biological tissue DRSdirectly measures the attenuation of an optical signal when light ofwavelength between 330 and 830 nm is emitted into the tissue. Whileabsorption is primarily due to the chromophores deoxy andoxy-hemoglobin, the scattering properties may be related to the size anddistribution of cells, organelles, and heterogeneous tissue structure,and are mainly affected by the collagen fibers of the stroma. Collagenfibers are about 2-3 μm in diameter which is composed of collagenfibrils about 0.3 μm. Scattering from collagen fibers is dominant in thevisible range. Near infrared spectroscopy (NIR) uses wavelengths from700-1000 nm and has a greater penetration depth than DRS depending onthe distance between source and detector fibers (3-5 mm for the probeused). DRS can only give information from 100-500 microns. Because ofthe penetration depth and the wavelength used in DRS, blood vesselorganization and presence of neutrophils is less a factor in DRSscattering as compared to collagen concentration. These two methodstherefore provide complementary information.

In the above experiments, the inventors were able to determine thedifference in the change of the absorption coefficient during the woundhealing period. In the control wounds, the rate of change in absorptioncoefficients (FIG. 4) was consistently higher at 685 nm, 780 nm, and 830nm compared to the diabetic wounds. This is the behavior predicted byvessel ingrowth in the control as blood vessel growth in the controlproceeded more rapidly. This was confirmed by image analysis of thetrichrome and lectin stains for vessel density. The scattering functiondetermined by DRS correlated very well with collagen concentrationdetermined by trichrome staining in both the diabetic and controlwounds.

In summary, absorption coefficients obtained using near infraredspectroscopy correlated with blood vessel ingrowth seen histologicallyand by vessel staining during healing and could differentiate thechronic wounds (e.g., diabetic, pressure ulcer, venous ulcer, ubiquitousulcer, and/or ischemic wounds) from the control (non-impaired ornormally healing) wounds and to identify pressure ulcers and/or venousulcers in the wound. Scattering function data obtained using DiffuseReflectance Spectroscopy (DRS) correlated with increasing collagenconcentration during the healing phase. The use of near infrared imagingof wounds may allow the clinician to assess normal wound healing anddevelop an optimal wound healing trajectory based on histologicalcorrelates. Active wound healing agents such as hyperbaric oxygen andtopical growth factors would be expected to shift the healing trajectoryof the impaired wound towards that of the normal. The data gatheringtechnique described above may be used to monitor values that may be, inturn, correlated to the healing state of the wound to, for example,enable a researcher to study the healing process and any mechanisms thatinterfere with the healing process. The healing state of the wound alsomay be used to determine whether any diagnosis or treatment arenecessary.

Deep Tissue Monitoring of Wounds

As noted above, diffuse reflectance spectroscopy (DRS) techniques may beused to collect data regarding wound tissue for depths up to 1 mm.However, by using Diffuse Photon Density Wave (DPDW) methodology of nearinfrared spectroscopy, one may further investigate tissue physiologyfrom a few millimeters up to several centimeters below the skin ortissue surface. Specialized instruments are built and operated at nearinfrared wavelengths (650-870 nm) where the tissue appears astransparent as possible to that light. At these wavelengths theabsorption coefficient μ_(a) of tissue is markedly lower than its valueat visible wavelengths. The propagation of light in tissue ischaracterized by three phenomena: scattering, absorption and reflectionfrom various layers. The diffusion equation can describe lightpropagation in tissue if the characteristic distance between successivephoton scattering events (mean free path) is much less than 1/μ_(a) butlarger than the wavelength of incident light. Then the dominantphenomenon of light propagation in tissue is multiple light scatteringby cells, organelles, capillaries, and other interfaces and tissuestructures. This is indeed the case at NIR wavelengths, where absorptionof hemoglobin, water and lipids is relatively very small (for hemoglobinless by a factor of 30-50 compared to absorption at 540-580 nm, therange used in DRS methodologies for determining blood oxygenation).Furthermore, at a selected range of NIR wavelengths, the spectra of oxyand deoxy hemoglobin are significantly different from each other andallow calculation of absolute concentrations of both types ofhemoglobin, and consequently, oxygen saturation, if their extinctioncoefficients at the particular wavelengths are known. For specialboundary conditions of the diffusion equation, simple closed formsolutions can be obtained that allow calculation of absorption andscattering coefficients at specific NIR wavelengths from experimentaldata.

The DPDW method can yield quantitative information about bloodoxygenation and blood volume, water and lipid content, as well asqualitative information about changes of tissue structure. There aremany biomedical applications where use of this non invasive opticalmethod be used to gather data that is, in turn, used by physicians todiagnose a wide range of medical pathologies. This includes cases whereblood supply to the tissue changes significantly as a result of thedisease, as in tumor angiogenesis. In stroke, aneurysm, or brain damageand head injury bleeding or ischemia can be determined by opticalmethods. Additional applications lie in the areas of hemodynamics ofhuman muscle, peripheral vascular diseases, control of photodynamictherapy (PDT) and monitoring of lesions.

The potential of using the DPDW methodology to characterize subcutaneouslesions and assess the necrotization depth of burns was discussed forthe first time in a paper by Tromberg et al. entitled “Reflectancemeasurements of layered media with diffuse photon-density waves: apotential tool for evaluating deep burns and subcutaneous lesions,”Phys. Med. Biol., 1999, Vol. 44(3), pp. 801-813. In a previous study bythe present inventors (Papazoglou et al., “Optical Properties of Wounds:Diabetic Versus Healthy Tissue,” IEEE Transactions on BiomedicalEngineering, 2006, Vol. 53(6), pp. 1047-1055), the use of DPDWmethodology at NIR wavelengths to distinguish the optical properties ofdiabetic wounds from normal wounds in an animal model was reported. Inthat study, the results of two new animal studies in which temporalchanges in the optical properties of wound and non-wound tissue aremonitored with DPDW methodology at NIR wavelengths throughout the courseof wound healing were reported. The absorption and scatteringcoefficients can be calculated, and blood oxygenation can be quantifiedby using the diffusion approximation with the semi-infinite boundarycondition. The inventors' approach has been to measure opticalproperties of the wound tissue in vivo and to calculate tissueoxygenation using the optical absorption coefficient. Since depthpenetration is accomplished at relevant physiological depths, there isno need for empirical fitting of spectroscopic data. The data obtainedfrom in vivo measurements taken in accordance with the inventionstrengthen and support the conclusions of Tromberg et al on theadvantages of using DPDW to study necrotic burn tissue or skin lesions.Differences in tissue optical properties between the wound andnon-wounded site during the course of healing can reveal informationabout physiological changes of the tissue, such as its inflammatorystate and its rate of healing. The results presented below indicate thatthis NIR method would be highly useful in collecting data whose valuesmay be used to monitor and quantify the wound healing process.

Materials and Methods

Optical Methods

A frequency domain DPDW instrument illuminated the animal tissue withfour diode lasers in the near infrared window at wavelengths of 685,780, 830 and 950 nm, with its intensity modulated by a radio frequencyω=70 MHz. A schematic of the device and a detailed description can befound in the afore-mentioned article by Papazoglou et al. entitled“Optical Properties of Wounds: Diabetic Versus Healthy Tissue,” IEEETransactions on Biomedical Engineering, 2006, Vol. 53(6), pp. 1047-1055.Backscattered light was delivered to four detector blocks based onAvalanche Photodiodes (APD) and quadrature (I/Q) demodulators. The I andQ signals in each detector were measured, and these were determined bythe attenuated amplitude A_(att) and phase shift Θ_(lag) of theregistered scattered light. The output power at the end of the sourcefiber ranged from 5 to 7 mW, for all four wavelengths.

The diffusion approximation can be used to calculate absorption μ_(a)and reduced scattering μ′_(s) coefficients of tissue based on thesolution of the time-dependent diffusion equation assuming asemi-infinite tissue geometry as described by Haskell et al. in“Boundary conditions for the diffusion equation in radiative transfer,”J. Opt. Soc. Am. A, 1994, Vol. 11(10), pp. 2727-2741, and by Pham et al.in “Broad bandwidth frequency domain instrument for quantitative tissueoptical spectroscopy,” Review of Scientific Instruments, 2000, Vol. 71,pp. 2500-2513. The closed form analytical solutions to the diffusionequation were used for calculating the optical properties of animaltissue using the techniques disclosed by Pham et al. The so-calledextrapolated condition for semi-infinite media was found to be a goodapproximation in non-invasive clinical applications where the fluencerate is nonzero at the boundary.

During this diffusion and “snake-like” propagation of light in thetissue, light is attenuated in intensity and also subjected to a phaseshift which reflects the mean flight time of photons through thestrongly scattering medium (tissue). The reduced scattering coefficientis defined as a function of the scattering coefficient μ_(s),μ_(s)′=μ_(s)(1−g), where the average cosine angle of scattering g˜0.9for biological tissue and its inverse is defined as the mean transportlength l*. Usually after propagation of more than two or three l*,photons have no memory of the incident direction of light and it can beassumed that the radiance is quasi-isotropic.

For most biological tissues μ′_(s) is between 5-15 cm⁻¹ and its valuedetermines the design of the appropriate experimental probe. In studiesconducted by the present inventors, it was assumed that μ′_(s)˜10 cm⁻¹for animal tissue and an optimal probe was designed. The scatteringcoefficient calculated from the present studies was very close to theassumed value of 10 cm⁻¹, corresponding to l* around 1 mm. Since thesmallest source detector separation of the probe (4 mm) used by theinventors is larger than 3*l*, the diffusion approximation will bevalid.

The optical fibers were inserted in a Teflon probe of length 25 mm andwidth 7 mm, with a separation between source and detector fibers of ρ=4,8, 12 and 16 mm. It is possible to estimate the probable penetrationdepth of diffuse light D_(v) in the tissue as function of thesource-detector separation ρ by using diffusion theory. A detailedinvestigation of this problem can be found in articles by Fridolin etal. entitled “Optical non-invasive technique for vessel imaging: II. Asimplified photon diffusion analysis,” Phys. Med. Biol., 2000, Vol.45(12), pp. 3779-92, and by Weiss et al. entitled “Statistics ofPenetration Depth of Photons Re-emitted from Irradiated Tissue,” Journalof Modern Optics, 1989, Vol. 36(3), pp. 349-359, but a rule of thumboften applied is that: D_(v)˜(1/3−1/2)ρ.

Calibration Procedures

The measured intensity of scattered light A_(att) depends not only onthe tissue properties, but also on the sensitivity of the AvalanchePhotodiode (APD), the coupling to the detectors fibers, the transmissionof the optical fibers and the gain of each detector block. The phaseshift Θ_(lag) may be different in each channel because the optical andelectrical signal delay depends on fiber length and coupling, the lengthof the RF coaxial cables, and any delays in the detector circuits.Instrument calibration is performed to allow separation of thevariability due to the instrument hardware components from sample andmeasurement variability.

An equidistant probe is constructed to conduct the first instrumentcalibration. The four detector fibers are inserted in a Teflon probewith the same source-detector separation of 12 mm. The probe is placedon the surface of a liquid optical phantom (Intralipid) that simulatestissue optical properties in a semi-infinite geometry. The set ofcalibration coefficients that equalizes the amplitude and phase of the2^(nd), 3^(rd) and 4^(th) detector relative to the 1^(st) detector isdetermined. All subsequent experimental data are corrected using thisset of calibration coefficients.

It should be noted that the use of an Intralipid solution as an opticalphantom for experiments that span several days is not the best approach,because the solution changes optical properties due to phase separationand degradation. An additional factor that contributes to operatorvariability when using Intralipid is the repeatability of placing thesolid plastic probe exactly on the surface of the solution. Use of thesemi-infinite approximation relies on perfect contact between the solidand liquid interface, without any air gap and also without immersing theprobe in the liquid. Solid phantoms can overcome some of thesechallenges. Silicone optical phantoms were the method of choice forcalibrating the NIR device because these models do not change opticalproperties during the time course of our experiments. Cylindricalphantoms made of silicone with dispersed particles of titanium dioxideto act as scatters and carbon black to act as an absorber were used.Cylinders with diameter of 90 mm and thickness of 45 mm were synthesizedfrom silicone XP565 with activator (platinum catalyzed from SiliconesInc), and TiO₂ particles with diameter between 0.9-1.6 μm simulatedtissue scattering and carbon black acetylene, 50% compressed, 99.9+%(metals basis) (diameter=0.042 μm) simulated light absorption. Both TiO₂and carbon black were obtained from Alfa Aesar.

The inventors optimized the preparation of these models includingintensity and time of mixing, the order of addition of the componentsand the crosslinking reaction to ensure phantoms of desired compositionwith no air bubbles. The absence of microbubbles was verified bysectioning the phantoms in thin layers and observing their surfacesunder an optical microscope.

Typical of any device that measures light intensity, the instrument hasa limited range where the electrical output signal is proportional tothe optical power of the input signal. A second calibration was thusconducted to define the region of saturation which occurs at an outputsignal of around 100 mV. The linearity range for the device used in thisstudy was >50 dB. Typical magnitudes of the I and Q demodulation signalswere in the range of 2-70 mV. Offset for the instrument, defined as thesignal measured without any light, was measured before every experimenton an animal and has not exceeded 500 μV for any experiment, with anaverage value around 250 μV throughout the studies. This calibrationexperiment allows one also to calculate the Noise-Equivalent Power (NEP)for the device, which was equal to 5 pW/Hz.

Animal Models

Hairless rats were used as the animal model for studying tissue opticalproperties during wound healing. This is a model that is widely used andaccepted for studying skin and wound properties. The absence of hairremoves the complications of inflammation introduced by shaving thewound site, and does not interfere with the optical measurements.

Two independent studies were performed as described below:

1^(st) STUDY: Three female hairless Sprague Dawley rats, 5-6 weeks oldand approximately 150 g each, were purchased from Charles RiverLaboratory (Wilmington, Mass.). A measurement protocol was developedover the course of 15 weeks, and when measurements began the ratsweighted approximately 300 g each. The rats were monitored with NIR for48 days (FIG. 12), with independent measurements taken usually every 3-4days. On the 48th day, one quarter-sized (4.6 cm²) full thickness wound(FIG. 13) was inflicted on the left dorsal area of each animal in orderto produce a wound animal model on all rats. A full thickness wound is asuperficial wound where the epidermis and dermis are removed to exposethe underlying tissue. It is different from an incision wound and itheals by contraction. Sixteen series of optical measurements wereperformed on the wound and on skin bordering the edge of the wound.Symmetrical measurements were performed on the right dorsal side of allanimals (FIG. 13).

2^(nd) STUDY: Twelve healthy rats identical to the ones in the firststudy were purchased and allowed to acclimate to their surrounding for 4weeks until they weighed approximately 200 g each. Baseline nearinfrared data were collected on all rats for 33 days (FIG. 12), withindependent measurements taken every 3-4 days. On day 36 a fullthickness wound 4.6 cm² was made using sterile technique in an animalsurgical suite. One wound was inflicted on the left side of the dorsalarea of each animal. The right side of each animal was left unwounded toprovide a control site. NIR measurements were performed on the woundsand control sites until day 57 (FIG. 13) when the wounds were completelyre-epithelialized. The wound surgery and all optical measurements wereperformed using isoflurane and oxygen anesthesia administered via facemask to prevent the animals from moving. It was necessary to anesthetizethe animals to eliminate motion artifacts before performing NIRmeasurements. Animals were measured as soon as they stopped moving andNIR measurements lasted 5 minutes at most. All wounds were covered witha Tegaderm (3M, Minneapolis, Minn.) sterile transparent dressing afterwound surgery and between optical measurements. After surgery all ratswere fitted with “Elizabethan” type collars to prevent them fromscratching their wounds.

Immunohistochemistry

Tissue Excision

During the second study, three rats were sacrificed by CO₂ suffocationon days 5 and 10 after wound surgery, respectively. The wound andsurrounding skin were completely excised, as was the area of the dorsumcontralateral to the wound. This procedure was repeated for theremaining 6 rats on day 21 after wound surgery. All excised tissue wasimmediately frozen at −80° C. until needed.

Blood Vessel Staining

Lectin staining (a sugar binding protein of non-immune origin thatagglutinates cells or precipitates glycoconjugates) was used to stainvessels in the tissue, and visualize vascularization. Lectin can be usedas a marker of angiogenesis because it binds to endothelial cells andreveals the overall vascular architecture. Briefly, sections were washedin 1×PBS for 10 minutes after rehydration. Sections were stained withAlexa Fluor 488 conjugated lectin (Invitrogen L2-1415) for 30 minutes inthe dark with a concentration at 1:250 and washed with 1×PBS 3 times for5 minutes each.

Results

Baseline

The stability and accuracy of the frequency domain NIR instrument usedin the study is demonstrated in FIG. 14, which tracks the absorption andreduced scattering coefficients over the course of 50 days measured insilicone phantoms. Standard error remained at less than 4% throughoutthe period of the study. Of course, those skilled in the art willappreciate that appropriate time domain NIR instruments may be used aswell.

In order to be able to detect the small changes in optical propertiesoccurring during wound healing the NIR device used should exhibit verygood stability. Otherwise it would be impossible to discern systematicdevice drift from actual physiological changes. The 48-day and 36-dayperiods of in vivo measurements prior to wound surgery in the 1^(st) and2^(nd) studies, respectively, allowed the inventors to determine withhigh consistency the local values of μ_(a) and μ′_(s) for the animals.

These values form the baseline measurements for assessing changes inoptical properties during the wound healing studies. Combined results ofbaseline measurements for μ_(a) and μ′_(s) from both animal studies areshown in FIG. 15 for 685 nm. The error bars in FIG. 15 indicate thebetween-animal variation, which was less than 15% percent of the averagevalues of μ_(a) and μ′_(s) for each time point in study 2. Similarresults were obtained for 785 nm and 830 nm measurements. Baselinemeasurements for three representative rats are presented in FIG. 16.Within-animal variation was less than 15% of the average values of μ_(a)and μ′_(s) for each animal in the second study.

It is noted that FIG. 15 illustrates the average absorption andscattering coefficients for all animals measured as a function of time.Baseline values of (a) left dorsal, (b) right dorsal, (c) left dorsal,and (d) right dorsal at 685 nm from study 1 and study 2 are illustrated.Each point represents the average of measurements taken on that day;error bars represent the standard deviation. In both studies, baselineoptical measurements were taken at symmetric locations on the left andright dorsa of each animal. The average data of baseline stabilityobtained during the 2nd study (black points) is a better indicator ofdevice stability because of the higher number of animals (n=12) comparedto n=3 in the first study (gray points).

FIG. 16 illustrates left dorsal baseline values of (a) and (b) fromthree representative rats. Each point represents the average of threemeasurements and error bars represent the standard deviation.

If the in vivo data is compared with those obtained from phantoms, it isclear that in addition to the noise from the laser-diodes, electronics,and fibers, being common to both in vitro and in vivo measurements,additional noise emanates from physiological changes in the animaltissue during the experiments. Several reasons may be responsible forsuch changes: The size of the rat is small, even compared to a 2 cmprobe. The rats were anesthetized during measurements and unable tomove; however breathing may have contributed to unintended change inprobe positioning. The food supply was provided ad libitum, and this mayhave affected the amount of blood at the measurement sites at varioustimes. Rats have been growing during the period of baseline measurementsand therefore a slightly different tissue volume was examined as timewent on.

Attention is drawn to the fact that absorption coefficient issystematically higher for the left dorsal side as compared to the rightone, for all animals at all timepoints (FIG. 15). During the 2nd studythe differences between the two sides range from 0.01-0.015 cm and thismay be due to asymmetry in the animal physiology.

Optical Properties During Wound Healing

In the experiments, wound size was determined by calculating woundsurface area from cross-polarized digital images, taken at the same timethe near infrared data was collected. The image analysis tool IMAGE PRO(Media Cybernetics, Silver Spring, Md.) was used to calculate the areaof each wound. Although the original size of each wound was largerelative to the size of the rat, the wound healing rate was very fastfor this model (as with all healthy animals) and evident of the intensephysiological changes in the animal during healing. A normalized woundarea was obtained by calculating the ratio of wound area each day to theinitial wound area on the day of surgery (day 0). Average normalizedwound areas are presented in FIG. 17 (Study 2). Wound healing rate inthis animal model exhibits a non-linear behavior as reported by Mast etal in “Optical Measurements of tissue oxygen saturation in lower limbwound healing,” Adv. Exp. Med. Biol., 2003, Vol. 540, pp. 265-9. Fromthe data, it can be observed that the healing rate is fast between days3-10 and then decelerates to achieve full wound closure. (Note that thedata points on days 0 and 3 are connected by a dashed line because fromMast et al. it is known that the rate exhibits highly non-linearbehavior prior to day 3.

The change of optical properties was monitored during wound healing forall animals. Changes of optical properties at 685 nm as a result of theexperiments are shown in FIG. 18. The absorption coefficient of thewound is increasing during wound healing and asymptotically approaches avalue that is higher by 0.035-0.040 cm⁻¹, or 35-40%, (FIG. 18, opensquares and triangles) compared to the non-wounded site (FIG. 18, filleddiamonds) throughout the experiment. The difference in μ_(a) betweenwound and non-wound tissue is statistically significant (p<0.01) afterday 5. The difference in μ′_(s) between wound and non-wound tissue isstatistically significant (p<0.05) after day 3. Similarly shaped healingcurves were observed at other wavelengths, with μ_(a) at 780 nmincreasing by 0.030-0.035 cm⁻¹ (approximately 35%) and μ_(a) at 830 nmincreasing by 0.040-0.045 cm⁻¹ (approximately 40%) when compared to thenon-wounded site.

In FIG. 18, (a) illustrates μ_(a) and (b) μ′_(s) at 685 nm during woundhealing (average ±standard deviation) for animals in study 1. Woundsurgery was performed on day 0. Open triangles represent measurementstaken on the edges of the wounds; open squares represent measurementstaken at the center of the wounds, and closed diamonds represent controlmeasurements on the non-wounded site. In FIG. 18, (c) μ_(a) and (d)μ′_(s) at 685 nm during wound healing (average ±standard deviation) foranimals in study 2. Again, wound surgery was performed on day 0. Opensquares represent measurements taken at the center of the wounds, andclosed diamonds represent control measurements on the non-wounded site.Two-tailed paired t-tests were performed to compare wound center andcontrol data at each time point (*p<0.01, **p<0.05).

Increasing values of μ_(a) as the wound is healing could be due toangiogenesis and neovascularization and this was supported byimmunohistochemical analysis where vessel ingrowth increased with timein lectin-stained images of blood vessels as shown in FIG. 19 forstained endothelial cells. FIG. 19 illustrates lectin-stained images ofwound tissue on (a) day 5, (b) day 10, and (c) day 21 after woundsurgery where the vascular structures are stained. The number and sizeof vascular structures increases as the wound heals.

As may be seen from FIG. 18, the values of μ_(a) obtained frommeasurements on the center of the wounds are identical (withinexperimental error) to the absorption coefficients obtained frommeasurements of the peri-wound area particularly for the first severalmeasurements when the wounds are large in size compared to the probe.This remained consistent in both animal studies, and can be explained bythe geometry of the experiments. Studies of photon penetration depth atthese wavelengths with geometry similar to the one used have shown thata probe having a source-detector separation of 16 mm registers scatteredlight from a tissue volume up to 5 mm beneath its surface (Weiss et al.,Statistics of Penetration Depth of Photons Re-emitted from IrradiatedTissue,” Journal of Modern Optics, 1989, Vol. 36(3), pp. 349-359. Thesimilarity between optical properties measured at the wound center andwound periphery provides evidence that the measured tissue is locatedbeneath the skin's surface, and therefore overlapping tissue volumes areinterrogated as the probe is positioned on the center or the peripheryof the wound. This observation may have clinical utility because itindicates that a wound could be monitored without the fiber optic probetouching directly the surface of an open wound.

The data of FIG. 18 suggest that during normal wound healing the opticalproperties of tissue at NIR wavelengths change measurably, and thereforehealing may be followed by measuring changes of the absorptioncoefficient of the wound. Oxyhemoglobin concentration ([HbO₂]) anddeoxyhemoglobin concentration ([Hb]) were calculated from the values ofμ_(a) and μ′_(s) using a modified form of the Beer-Lambert equation:

ε_(Hb) ^(λ)[Hb]+ε_(HBO2) ^(λ)[HbO₂]+μ_(a,H2O) ^(λ)[% H₂O]=μ_(a,measured)^(λ)  (1)

where ε_(Hb) ^(λ) and ε_(HBO2) ^(λ) are the molar extinctioncoefficients of deoxy- and oxyhemoglobin, μ_(a,H2O) ^(λ) is theabsorption coefficient of pure water, and [% H2O] is the percentage ofwater in the measured tissue, which is assumed to be 70%. Meanhemoglobin values increased during wound healing, as shown in FIG. 20.Within the accuracy limits of the experiment, no significant change inoxygen saturation was obtained during the course of wound healing, asshown in FIG. 21. Oxygen saturation is defined as,

${{SO}_{2} = \frac{\left\lbrack {{Hb}O}_{2} \right\rbrack}{\left\lbrack {{{Hb}O}_{2} + {Hb}} \right\rbrack}},$

where HbO₂ and Hb are the concentrations of oxygenated and deoxygenatedhemoglobin.

This small change supports the findings by T. K. Hunt et al. in “Oxygenand wound healing,” Hyperbaric Medicine 2000, 8^(th) Annual AdvancedSymposium, 2000, and Jonsson et al. in “Tissue oxygenation, anemia, andperfusion in relation to wound healing in surgical patients, Ann. Surg,1991, Vol. 214(5), pp. 605-613, who suggested that oxygen saturation isnot a sensitive measure of wound healing because hemoglobin delivery tothe wound environment is disrupted by microvasculature damage,vasoconstriction, and clotting in the area surrounding a wound. However,the optical properties of tissue change measurably in this animal modelduring wound healing in contrast to the insignificant change of oxygensaturation. Therefore, tissue absorption coefficients may have adequatesensitivity to be good global indicators of changes during woundhealing.

Discussion

In the NIR region, the change of the absorption coefficient μ_(a)reflects the variation in oxygenated and deoxygenated hemoglobinconcentration because hemoglobin is the main absorption chromophore atthe wavelength range 680-870 nm along with water and lipids. The NIRabsorption coefficient during wound healing (FIG. 18) increases on thewound side by 0.020-0.035 cm⁻¹, and total hemoglobin concentrationincreases by 0.06-0.07 mM (FIG. 20). This means that during normalhealing the optical properties of tissue change measurably in thisanimal model as a result of a 30-35% difference in blood volume betweenthe wound side and the control side. It would be important to monitorhow the absorption coefficient returns to normal levels (pre-wound)after the tissue has remodeled fully and the system recovered from thewound perturbation.

The experimental results demonstrating baseline differences μ_(a)between the left and right dorsal side highlight the importance ofselecting a control site with well-understood optical propertiesrelative to the wound site, and that a contralateral position may not bethe optimal control site. In a clinical application, opticalmeasurements occur on patients with already existing wounds. Therefore,trends of absorption and scattering coefficients of the wound sitesshould be looked at over time. Since it will be not be possible tocompare the optical properties of patient wounds to any pre-woundbaseline, in the clinic it is desirable to select a control site withstable optical properties. The optical properties of this control sitewill be used to establish the baseline stability of the human studydescribed below. In the framework of the experimental model, theabsorption coefficient should decrease if proper healing is occurring,as demonstrated in FIG. 22, which illustrates a hypothesized clinicalwound healing curve. The dark solid lines represent the result of theanimal study shown in FIG. 18, while the other lines are hypothesizedcurves for healing and non-healing wounds. The amount of time requiredfor the healing curve to converge to the baseline is not known.

In order to further analyze the experimental results, two-tailed t-testswere performed to understand how the optical data reflect the process ofwound healing in this animal model. The t-test allows differentiation ofvalues of absorption and scattering over time with statisticalsignificance and finer detail compared to a simple comparison of averagevalues and their standard deviations. Absorption and scatteringcoefficients from day 3 of wound healing were compared to data from eachsubsequent timepoint using t-tests. The results, presented in FIG. 23,show that the p-value obtained for the 685 nm absorption coefficientbecomes very small (at the level of 0.01) at day 18 for the wound centerand at day 8 for the wound edge. FIG. 23 illustrates two-tailed,unpaired t-tests that were used to compare the average opticalcoefficients at each time point to the average optical coefficients onday 3. The resulting p-values are shown as a function of time for (a)μ_(a) at center of wound, (b) μ_(a) at edge of wound, (c) μ′_(s) atcenter of wound, and (d) μ′_(s) at edge of wound. The absorption at 685nm is due mostly to deoxygenated hemoglobin corresponding to the tissuemetabolic activity. Therefore, at the wound center there may be a timelag for significant metabolic activity. At the other two wavelengths 785nm and 830 nm, the p-values are systematically higher demonstrating that685 nm absorption may be a more sensitive indicator of metabolic changesthat occur during wound healing than absorption at other wavelengths andoxygen saturation. Another very important conclusion from these data isthat the p-value decreases earlier for the wound edge than it does forthe wound center. These results are in agreement with a healing woundmodel where healing starts from the edges and the wound heals bycontraction. This is the wound healing mode followed by this animalmodel where healing starts from “around” the wound, with increasedmetabolic activity and the wound center is the last location whereepithelialization (new skin) is formed.

The demonstrated baseline stability of the device makes possible to usethis method in a clinical setting where measurements are performed onchronic wounds spanning periods of 6-12 months. The results suggest thatthe NIR methodology and instrument developed by the inventors is stableand capable of detecting changes to optical properties connected towound healing. This quantitative non-invasive method could complementthe current practice of monitoring wound healing based on visualobservation and measurement of wound size to improve the quality ofwound care, particularly for chronic wounds due to diabetes, pressureulcers (bed sores), venous ulcers, ubiquitous ulcers, ischemia, etc.Moreover, the method of the invention permits the identification ofpressure ulcers, venous ulcers, and the like that would not otherwise bevisible to upon visual examination of the wound.

In summary, the in vivo studies using the hairless rat animal model havedemonstrated that the absorption coefficient of tissue at all NIRwavelengths probed (680,785, 830) is higher in the wound compared withthe unwounded side of animals, corresponding to increasedvascularization. The observed differences in μ_(a) between the woundedand unwounded side of animals can be attributed to the traditionalchromophores of oxygenated and deoxygenated hemoglobin, because noevidence of a different type of tissue chromophore in these wavelengthswas found. The results also demonstrate that the right and left side ofthese animals are slightly asymmetric in their optical properties andthis should be further explored for long term wound healing studies. Asin the previous embodiment, the data gathering technique described abovemay be used to monitor values that may be, in turn, correlated to thehealing state of the wound to, for example, enable a researcher to studythe healing process and any mechanisms that interfere with the healingprocess. The healing state of the wound also may be used to determinewhether any diagnosis or treatment are necessary.

Human Data Materials and Methods

Near Infrared Instrumentation

A frequency domain near infrared instrument of the type described byPapazoglou et al. in “Optical properties of wounds: diabetic versushealthy tissue,” IEEE Trans. Biomed. Eng., 2006, 53(6), pages 1047-55,was used. An optical fiber was used to deliver intensity modulated light(70 MHz) to the tissue from three diode lasers (λ=685, 780, and 830 nm).Four optical fiber bundles were used to deliver backscattered light fromthe tissue to avalanche photodiode (APD) detectors and quadrature (I/Q)demodulators. The I and Q signals in each detector were measured; thesewere determined by the attenuated amplitude and phase shift of theregistered scattered light. All optical fibers were immobilized on aTeflon probe, with the four detector fibers fixed at distances of 4, 8,12, and 16 mm from the source fiber.

It is known by those skilled that absorption and scattering coefficientsof tissue may be calculated from the amplitude and phase shift ofscattered NIR light using the diffusion approximation if the probe has aminimum distance between source and detector fibers greater than acouple transport mean free paths. The transport mean free path (l*)represents the distance of propagation of a collimated beam of lightbefore it becomes effectively isotropic, and can be approximated by1/μ_(s)′ when μ_(s)′>>μ_(a), as is the case in tissue. After propagatingmore than approximately a couple transport mean free paths, most photonshave undergone multiple light scattering (i.e., they are now at adifferent orientation from their incident direction) and may bedescribed as diffuse. Values of μ_(s)′ in human skin at wavelengths of685-830 nm typically range from 5-20 cm⁻¹; therefore, the transportlength l* ranges from approximately 0.5 to 2 mm, since l* is the inverseof the reduced scattering coefficient μ_(s)′. This suggests that thesmallest source-detector distance that can be used in probe design forthe diffusion approximation to be valid would be 2-4 mm. The probe usedby the inventors has a minimum distance between source and detectorfibers of 4 mm and therefore is within the diffusion approximationregime. Closed analytical solutions to the diffusion equation have beenderived for semi-infinite measurement geometries that are typical ofnoninvasive tissue measurements, when sources and detectors are placedon an air-tissue interface and the optical fiber source is modeled as anisotropic, point light source. The final equations describing theabsorption and scattering coefficients from measurements of lightintensity and phase shift as a function of the source-detectorseparation distance are included in the afore-mentioned article byPapazoglou et al.

The human study lasted for over a year and it was therefore necessary totest the stability of the device during the course of such measurements.To accomplish this, an optical phantom made of silicone(XP565—Silicones, Inc.) with dispersed particles of TiO₂ (diameter 0.9to 1.6 μm—Alfa Aesar) to act as scatterers and carbon black acetylene(50% compressed, diameter 0.042 μm—Alfa Aesar) to absorb light wasmeasured before each patient measurement session. The measuredabsorption coefficients from the silicone phantom over the course of 61weeks are shown in FIG. 24. In particular, FIG. 24 illustrates dailyaverage values of μ_(a) in a silicone optical phantom over a 61-weekperiod. Each point represents the average of measurements taken on thesame day. Solid lines represent average values for the entiremeasurement period. Standard error remained at less than 3% throughoutthe period of the study.

Oxyhemoglobin concentration ([HbO₂]) and deoxyhemoglobin concentration([Hb]) were calculated from the measured values of μ_(a) by minimizingthe difference between expected and measured absorption of tissue atthese wavelengths (the left and right sides of the following equation):

ε_(Hb) ^(λ)[Hb]+ε_(HBO2) ^(λ)[HbO₂]+μ_(a,H2O) ^(λ)[% H₂O]=μ_(a,measured)^(λ)  (1)

where ε_(Hb) ^(λ) and ε_(HBO2) ^(λ) are the molar extinctioncoefficients of deoxy- and oxyhemoglobin, μ_(a,H2O) ^(λ) is theabsorption coefficient of pure water at each wavelength (λ), and theconcentration of water [% H₂O] was assumed constant at 70%. The choiceof a value for [% H₂O] has little effect on the calculated values ofhemoglobin concentration because of the low absorption of water relativeto hemoglobin at wavelengths in the range 685-830 nm. Total HemoglobinConcentration [Tot Hb] was calculated as the sum of [Hb] and [HbO₂].

Human Subjects

Eleven subjects with diabetes and chronic wounds were recruited from theDrexel University Wound Healing Center in Philadelphia, Pa. All patientswere between 18 and 65 years of age, had documented diabetes mellitusfor at least 6 months, and had an ankle or foot wound with a minimumsurface area of 1 cm² that was secondary to the complications ofdiabetes, including vascular disease and/or neuropathy. All patientsreceived standard wound care, which included weekly or biweeklydebridement, treatment with moist wound healing protocols, andoffloading when appropriate. In some patients, active wound healingagents such as topical growth factors, hyperbaric oxygen, andbioengineered skin substitutes were employed. Details about the size ofeach wound, duration of measurements, and the active treatments used oneach wound are shown in Table 1.

TABLE 1 Size, duration, and active treatments used on each wound InitialFinal Number Wound area area of ID (cm²) (cm²) weeks Active treatmentHealing 6.1 0.1 10 topical growth factor (Regranex) #1 Healing 1.2 0.010 hyperbaric oxygen #2 Healing 4.5 0.0 14 topical growth factor(Regranex) #3 Healing 4.5 0.2 14 bioengineered skin substitute #4(Apligraf) Healing 5.6 0.0 12 topical growth factor (Regranex) #5 Non-17.4 10.3 36 bioengineered skin substitute healing #1 (Dermagraft) Non-50.0 21.5 13 none healing #2 Non- 15.6 11.3 30 hyperbaric oxygen healing#3 Non- 14.1 3.2 61 topical growth factor (Regranex) healing #4 Non-74.5 17.7 16 hyperbaric oxygen healing #5 Non- 31.5 13.8 32 hyperbaricoxygen healing #6 Non- 16.2 4.5 15 hyperbaric oxygen healing #7

Of the 11 wounds enrolled in the study, five wounds healed completely inless than 15 weeks, three wounds resulted in amputation, and threewounds remained unhealed at the end of the study, as shown in Table 1.Four of the five healed wounds required no surgical intervention priorto closure, while one wound underwent surgical debridement and theapplication of a bioengineered skin substitute (Apligraf®,Organogenesis, Inc., Canton, Mass.) after 18 weeks of participation inthe study and reached closure after an additional 17 weeks. Dataobtained prior to surgical intervention were classified as a non-healingwound, while data obtained after surgery were classified as a healingwound, bringing the total number of wound to 12 (5 healing and 7non-healing).

All diffuse NIR measurements were conducted prior to wound debridementon a weekly or biweekly basis. During each measurement session, thewounds of each patient were interrogated using the NIR instrument in upto ten different locations. Measurement locations were chosen based onthe geometry and size of each wound, and can be classified into fourgeneral locations: (1) directly on the wound, (2) on intact skin at theedge of the wound, (3) on non-wound tissue of the contralateral limbsymmetric to the wound location if available, (4) on non-wound tissue onthe ipsilateral limb at a distance of at least 2 cm from the wound. TheNIR measurement locations for a typical diabetic foot ulcer are shown inFIG. 25. The dark oval on the heel of the right foot represents atypical diabetic foot ulcer. Gray rectangles represent the probelocations during a measurement session. Tegaderm transparent steriledressing (3M Health Care) was used to cover the fiber optic probe duringall measurements. The presence of Tegaderm has been found by theinventors to not affect the measured NIR coefficients.

Wounds were digitally photographed using a Fujifilm Finepix s700 digitalcamera during each measurement session with cross-polarizing filters toreduce surface reflection. Wound areas were calculated from thephotographs using an image analysis code developed with Matlab(Mathworks, Inc.) software.

Results

Results from Diabetic Foot Ulcers

In both healing and non-healing wounds, values of the NIR absorptioncoefficient μ_(a) at the wound center and wound edges were greater thanvalues of μ_(a) at the control (non-wound) sites. In all healing woundsthe values of _(μaat) the wound center and edge sites decreased andconverged to the values measured at the control sites. This isillustrated in FIG. 26, which shows plots of μ_(a) at all wavelengthsduring the course of the study for a typical healing wound. FIG. 26illustrates wound size, optical absorption, and hemoglobin data for arepresentative healed wound. In the upper left, digital photographs fromselected time points are illustrated. In the upper right, wound area asdetermined through analysis of digital photographs (♦). In the lowersections, mean values of μ_(a) at 685 nm, 780 nm, and 830 nm areprovided from each measurement day. Each data point represents the meanof measurements obtained from the center of the wound (), the edges ofthe wound (Δ), a control site on the wounded foot (+), and a controlsite on the non-wounded foot (x). The area of this wound, which waslocated on the frontal region of a foot that had previously lost alltoes to amputation, was over 6 cm² at the beginning of the study, andclosed after ten weeks of monitoring. In contrast, values of μ_(a) inall non-healing wounds remained greater than the control sites and didnot converge over the course of the study. This is illustrated in FIG.27, which shows plots of μ_(a) for a typical non-healing wound with thesame feature layout as in FIG. 26. A suitable control site on thewounded foot was unavailable due to the size of the wound and prioramputations. The area of this wound, which was located on the plantarmetatarsal region of the foot, decreased by only approximately 50% overthe course of 37 weeks, after which a below-the knee amputation wasperformed. FIG. 28 shows plots of μ_(a) for a unique case in which thewound initially appeared to be healing, decreasing in size from 31.5 cm²to 1.6 cm² over 17 weeks. FIG. 28 has the same feature layout as inFIGS. 26 and 27 but a suitable control site on the non-wounded foot wasunavailable due to prior amputations. However, this wound, which waslocated on the plantar metatarsal region of the foot, never closedcompletely and surgical intervention was required after week 25,increasing the wound size to 17.3 cm². Despite a rapid decrease in woundsize during the initial 17 weeks of the study, the NIR data from thewound site did not show convergence with the non-wound data as ischaracteristic of healing wounds in this study. This may indicate thatthe greater penetration depth achieved by diffuse NIR could provideclinicians with better assessment of wound status than superficialmeasurements of wound size.

The calculated values of total hemoglobin concentration [Tot Hb] for thewounds in FIGS. 26-28 are shown in FIG. 29. In FIG. 29, mean values oftotal hemoglobin concentration from each measurement day are illustratedfor healing wound #1 (left), non-healing wound #1 (center), andnon-healing wound #2 (right). Each data point represents the mean ofmeasurements obtained from the center of the wound (), the edges of thewound (Δ), a control site on the wounded foot (+), and a control site onthe non-wounded foot (x). As expected, the hemoglobin concentrationtrends are similar to those observed for optical absorption.

Rates of Change in Optical Data

In order to analyze the clinical data, the inventors identifies commonparameters that describe the observed trends and that are representativeof the clinical outcomes. In particular, the rate of temporal change ofthe absorption coefficient at each wavelength as well as the rate oftemporal change in hemoglobin concentration can be estimated by fittingthe data from each wound to a linear trend line. The limited amount ofexperimental data collected during this study combined with the dataaccuracy did not allow use of a more complicated fitting model at thistime. The slopes of the trend lines were found to correspond to therates of change in optical properties with time, and have proven usefulin quantifying the progress of a healing wound. The slopes calculatedfrom the optical absorption and hemoglobin concentration trend lines arereferred to herein as the rates of change in each wound, and are shownin FIGS. 30 and 31, respectively. Rates of change in optical absorptionfor all wounds are shown in FIG. 30 at wavelengths left: 685 nm, center:780 nm, and right: 830 nm. Similarly, rates of change in hemoglobinconcentration for all wounds are shown in FIG. 31 at Left: Totalhemoglobin concentration, center: Oxy-hemoglobin concentration, andright: Deoxy-hemoglobin concentration. White bars represent data fromthe centers of healing wounds; light gray bars represent data from theedges of healing wounds; dark gray bars represent data from the edges ofnon-healing wounds; black bars represent data from the centers ofnon-healing wounds. In all healing wounds negative rates of change wereobserved for the optical absorption coefficient at each wavelength, thetotal hemoglobin concentration, and the oxy-hemoglobin concentration. Inall non-healing wounds the rates of change for the above properties wereclose to zero or slightly positive. The rate of change fordeoxy-hemoglobin concentration was close to zero in both healing andnon-healing wounds. The mean rates of change in healing and non-healingwounds are compared for all optical absorption coefficients (μ_(a) at685 nm, 780 nm, and 830 nm) and all hemoglobin concentrations ([Tot Hb],[HbO₂], and [Hb]) in FIG. 32. In FIG. 32, error bars represent standarddeviation. One-tailed, heteroscedastic t-tests were used in FIG. 32 totest the difference between the rates of change in healing andnon-healing wounds, where *p<0.05 **p<0.01. As illustrated in FIG. 32, astatistically significant difference between the slopes of healing andnon-healing wounds was obtained for the optical absorption coefficientsat each wavelength, the total hemoglobin concentration, and theoxy-hemoglobin concentration.

Statistical Characterization of Healing and Non-Healing Wound Data

In addition to the rate of change of optical properties, the statisticalcharacteristics of optical data from a wound may be an indicator ofhealing potential. Visual comparison of FIGS. 26-28 reveals moreweek-to-week variability in the non-healing data than in the healingdata. To quantify variability differences, the root mean squaredeviation (RMSD) of experimental data from the fitted first-orderpolynomials was calculated. The RMSD values were normalized by dividingby the mean of the experimental values for each wound. FIG. 33 comparesthe mean values of normalized RMSD in healing and non-healing woundsusing the optical absorption at each wavelength and all hemoglobinconcentrations (oxy, deoxy and total). In FIG. 33, normalized RMSD ofthe lines are fitted to optical absorption (left) and hemoglobinconcentration (right) data. The mean normalized RMSD was greater innon-healing wounds than in healing wounds for all absorptioncoefficients and hemoglobin concentrations.

Discussion

The inventors have developed a hypothesized model of healing based onchanges in the NIR optical properties of wounds related tovascularization that were verified by histopathology andimmunohistochemistry. Specifically, results from the animal studiesdemonstrated increased optical absorption as blood volume increased inhealing wounds. The inventors further hypothesized that if DPDWmeasurements were continued after closure of the wound, a decrease inoptical absorption would be expected during the late proliferative phaseof wound healing as vessel density/blood volume decreases to normallevels. This decrease have been confirmed in absorption through anunpublished pilot study of wound healing in seven obese Zucker DiabeticFatty (ZDF) rats in which the wounds were monitored using DPDWmethodology throughout the healing cycle and for 42 days after woundclosure. The measured optical absorption coefficients at 830 nm behavedas expected, increasing prior to wound closure at day 33, and thengradually decreasing for the remaining 42 days of the study, as shown inFIG. 34. Each data point in FIG. 34 represents the mean of measurementsobtained from the center of the wound (, black line) and the edges ofthe wound (Δ, gray line).

A hypothesized model of the optical changes observed during woundhealing is illustrated in FIG. 35. In FIG. 35, the dotted black linerepresents normal (non-wound) tissue, while the descending dashed blacklines represent the hypothesized curve for healing wounds. The graymarkers represent measurements on non-healing wounds. The timedependence of NIR optical absorption for human patients is expected tobe different from that observed during the animal studies. Humanpatients are first seen when they have already developed chronic wounds,corresponding to an elevated yet constant absorption level (indicativeof non-healing) in the healing model. Any progress in healing manifestsitself by a decrease in the NIR absorption coefficient, and aconvergence to the value of non-wound tissue (FIG. 35, dashed blacklines). In wounds that do not heal, the level is not expected toconverge with non-wound tissue (FIG. 35, gray markers). The rates ofchange in healing and non-healing wounds summarized in FIG. 32 are inagreement with the hypothesized healing model and may provide the basisfor a quantitative “healing index” that helps clinicians to distinguishhealing from non-healing wounds.

The predictive capability of a quantitative healing index derived fromDPDW data may be confirmed through a study of more patients withmeasurements taken at more time points. As a first approximation, therate of temporal change of [Tot Hb] in each wound was calculated byfitting the data from the first 10 weeks of measurements to a lineartrend line. The slopes of the 10-week trend lines are compared to theslopes calculated from all available data in FIG. 36. In FIG. 36, ratesof change in total hemoglobin concentration from the centers of allwounds are illustrated. Dark bars represent rates calculated using allavailable measurements; light bars represent rates calculated using thefirst 10 weeks of measurements available for each wound. If −0.003cm⁻¹/wk is considered to be the threshold between a negative rate ofchange and a negligible rate of change, such that a negative rate ofchange predicts healing while a negligible rate of change predictsnon-healing, 100% of the healing wounds are correctly classified (4 of4) and 71% of the non-healing wounds are correctly classified (5 of 7).It is likely that the period of time needed to establish a predictivetrend could be reduced if measurements were conducted more frequently.[Tot Hb] trends are evaluated using 10 weeks of data because on averageeach wound was measured 5.0 times during its first 10-week period, and 5measurements appear to be adequate to establish a statistically adequatetrend line. If DPDW measurements were conducted every week, it might bepossible to establish a predictive trend line in only 5 weeks.

The rate of change of oxygenated hemoglobin concentration in healingwounds is greater than the rate of change in deoxygenated hemoglobinconcentration, as shown in FIG. 31. During the late inflammatory/earlyproliferation stage of wound healing, angiogenesis increases the supplyof oxygenated blood to the wound resulting in increased values of[HbO₂]. In the late proliferation stage, angiogenesis stops and bloodvessels begin to break down as a result of apoptosis. The resultingdecrease in supply of oxygenated hemoglobin to the wound may bereflected by the negative rate of changes of [HbO₂] observed in FIG. 31.Concentrations of deoxygenated hemoglobin reflect metabolic activitywithin the wound bed, and would be expected to remain relativelyconstant assuming that an adequate supply of oxygenated blood is beingdelivered to the wound. This could explain why the changes in [Hb] inhealing wounds were less pronounced than changes in [HbO₂].

It has been hypothesized that chronic wounds (e.g., diabetic, pressureulcer, venous ulcer, ubiquitous ulcer, and/or ischemic wounds) may be“stuck” in various phases of the healing process. The impaired(non-healing) wounds represented in FIGS. 27 and 28 may have been“arrested” before reaching the end of the proliferative phase ofhealing, resulting in oxygenated hemoglobin concentrations that werealways greater than normal tissue and did not decrease like non-impaired(healing) wounds. Furthermore, there is evidence that neuropathy and aprolonged inflammatory response in diabetic patients are importantfactors in the etiology of diabetic foot ulcers. Diabetic neuropathy isassociated with microcirculatory dysfunction in the foot, even inpatients who have normal large-vessel blood flow to the foot. It hasbeen hypothesized that repeated ischemia and reperfusion within themicrovasculature of the foot may lead to cycles of inflammation in footulcers, further impairing the wound healing process. The high degree ofweek-to-week variability in non-healing wounds compared to healingwounds shown in FIG. 33 supports this hypothesis and could be anindication of cyclical changes in the microcirculation and inflammatorystatus of the wound.

In summary, temporal changes in the NIR optical properties of diabeticfoot ulcers related to hemoglobin concentration can be measured usingthe techniques of the invention. Changes in the measured values may beused to monitor healing progress over time. These changes can bequantified by calculating the linear rate of change and the week to weekvariability in optical absorption coefficient and hemoglobinconcentration over time. These metrics were used to distinguish healing(non-impaired) from non-healing (impaired) wounds in a study of humandiabetic foot ulcers, indicating that DPDW methodology at near infraredwavelengths may be able to provide wound care clinicians with objectiveand quantitative data to help in the assessment of overall wound healthwhen deciding on treatment options. In other words, the overall woundhealth may be used to determine whether any treatment is necessary. Thenature of the treatment will depend on a number of factors including thenature of the wound, whether the patient is diabetic, the rate ofhealing of the wound, etc.

Those skilled in the art will appreciate that the near infrared (NIR)methodology disclosed herein probes tissue below the skin/wound surfaceat distances that depend upon the source fiber-detection fiber distanceand range from 2 mm to several cm of tissue depth. Therefore, theoptical properties of tissue can be assessed as well as the oxygenatedand deoxygenated hemoglobin concentrations, and hence tissue oxygensaturation. This allows the method to be used in a variety of ischemicenvironments, caused by problems in blood supply or problems in oxygensupply to the affected area. All cases of impaired healing andsubsurface compromised circulation can be assessed by the disclosedmethodology. For example, emerging pressure ulcers (bed sores) orubiquitous ulcers represent environments and tissue conditions that canbe assessed by the NIR method. Surface imaging may not reveal problemsunderlying a pressure or bed ulcer before it surfaces, but impairedsupply of blood and or oxygen which precedes such conditions can benon-invasively assessed by the NIR methodology described herein.

Those skilled in the art also will readily appreciate that manyadditional modifications are possible in the exemplary embodimentswithout materially departing from the novel teachings and advantages ofthe invention. For example, it is possible that modified simpler (e.g.,continuous wave) or more complicated (e.g., time resolved) methods ormodulation at higher frequencies of the frequency domain instrument canprovide similar information as provided using the techniques describedabove. These and other obvious extensions are also included within thescope of the invention. Accordingly, any such modifications are intendedto be included within the scope of this invention as defined by thefollowing exemplary claims.

1. A method of collecting information regarding the healing state of a wound, comprising the steps of: illuminating wound tissue with light from a light source; measuring the amplitude and/or phase shift of the light as it propagates through the wound tissue; calculating an optical absorption coefficient and/or reduced scattering coefficient using the measured values; correlating collagen concentration in the wound tissue with the reduced scattering coefficient calculated form measured parameters and/or correlating blood vessel in-growth and/or ischemia in the wound tissue with the optical absorption coefficient using the measured values; and determining changes in collagen concentration over time from changes in the reduced scattering coefficient over time and/or determining changes in blood vessel in-growth and/or ischemia over time from changes in the optical absorption coefficient over time.
 2. (canceled)
 3. The method of claim 1, wherein the light is transmitted at a near infrared wavelength.
 4. The method of claim 3, wherein the light has a wavelength of 650-870 nm.
 5. The method of claim 3, wherein the near infrared wavelengths comprise one of 685 nm, 780 nm, 830 nm, and 950 nm.
 6. The method of claim 3, wherein the light is output by a laser and modulated to produce a diffuse photon density wave (DPDW) in the wound tissue.
 7. The method of claim 1, further comprising measuring size of the wound by calculating wound surface area.
 8. The method of claim 7, further comprising measuring a healing rate of the wound by calculating the difference between the surface area of the wound at different points in time and dividing the difference by the original surface area of the wound.
 9. The method of claim 1, wherein correlating the collagen concentration in the wound tissue with the reduced scattering coefficient includes correlating an increase in a diffuse reflectance spectroscopy scattering function obtained over time in the wound with an increase in collagen during healing of the wound.
 10. The method of claim 1, further comprising measuring collagen concentration in the wound over time by taking diffuse reflectance spectroscopy (DRS) measurements over time.
 11. The method of claim 1, further comprising differentiating an impaired wound from a non-impaired wound by measuring changes in blood vessel in-growth and/or ischemia in the wound over time and correlating optical absorption coefficients obtained from the wound over time with blood vessel in-growth and/or ischemia seen histologically for impaired and control wounds.
 12. The method of claim 11, further comprising detecting pressure ulcers or venous ulcers in the wound from changes in the optical absorption coefficients over time.
 13. The method of claim 1, wherein the illuminating and measuring steps are performed using a continuous wave, a frequency domain, or a time domain measurement device that does not contact the wound.
 14. The method of claim 1, wherein the calculation of the optical absorption coefficients and/or reduced scattering coefficients and a quantification of blood oxygenation is performed using a diffusion equation form semi-infinite media.
 15. The method of claim 1, further comprising monitoring changes in oxygenated hemoglobin over time as an indication of whether the wound is healing.
 16. The method of claim 15, further comprising quantifying the changes in oxygenated hemoglobin by calculating a rate of change and variability in the optical absorption coefficient and hemoglobin concentration over time.
 17. The method of claim 16, further comprising differentiating a healing chronic wound from a non-healing chronic wound using rates of change of the oxygenated hemoglobin concentration over time.
 18. The method of claim 17, wherein the chronic wound is a diabetic ulcer.
 19. The method of claim 16, further comprising determining that the wound is healing based on convergence of absolute oxygenated hemoglobin concentration at a site of the wound to a value of absolute oxygenated hemoglobin concentration at a site of non-wounded tissue of a same patient having the wound.
 20. The method of claim 16, further comprising differentiating a healing chronic wound from a non-healing chronic wound using rates of change of total hemoglobin concentration over time.
 21. The method of claim 16, further comprising determining that the wound is healing based on convergence of absolute total hemoglobin concentration at a site of the wound to a value of absolute total hemoglobin concentration at a site of non-wounded tissue of a same patient having the wound. 